This invention relates to compounds which are useful for inactivating pathogens in a material, such as a blood product, and to methods of use of the compounds.
The transmission of disease by blood products and other biological materials remains a serious health problem. While significant advances in blood donor screening and blood testing have occurred, viruses such as hepatitis B (HBV), hepatitis C (HCV), and human immunodeficiency virus (HIV) may escape detection in blood products during testing due to low levels of virus or viral antibodies. In addition to the viral hazard, there are currently no licensed tests to screen for the presence of bacteria or protozoans in blood intended for use in transfusions. The risk also exists that a hitherto unknown pathogen may become prevalent in the blood supply and present a threat of disease transmission, as in fact occurred before the recognition of the risk of HIV transmission via blood transfusions.
Exposure of laboratory workers to blood or other body fluids also presents a health hazard. Twelve thousand health-care workers whose jobs involve exposure to blood are infected with hepatitis B virus each year, according to estimates from the Centers for Disease Control (xe2x80x9cGuidelines for Prevention of Transmission of Human Immunodeficiency Virus and Hepatitis B Virus to Health-Care and Public-Safety Workers,xe2x80x9d Morbidity and Mortality Weekly Report, vol. 38, no. S-6, June 1989).
Several methods have been proposed to complement donor screening and blood testing to decrease the incidence of disease due to transfusions. The introduction of chemical agents into blood or blood plasma has been suggested to inactivate pathogens prior to clinical use of the blood product. Nitrogen mustard, CH3xe2x80x94N(CH2CH2Cl)2, was added to blood components in an investigation of potential virucidal agents. However, substantial hemolysis occurred at the concentrations necessary to inactivate one of the viruses studied, rendering nitrogen mustard unsuitable for use in blood. LoGrippo et al., Proceedings of the Sixth Congress of the International Society of Blood Transfusion, Bibliotheca Haematologica (Hollander, ed.), 1958, pp. 225-230.
A xe2x80x9csolvent/detergentxe2x80x9d (S/D) method for inactivating viruses was described in Horowitz et al., Blood 79:826 (1992) and in Horowitz et al., Transfusion 25:516 (1985). This method utilized 1% tri(n-butyl)phosphate and 1% Triton X-100 at 30xc2x0 C. for 4 hours to inactivate viruses in fresh frozen plasma. Piquet et al., Vox Sang. 63:251 (1992), used 1% tri(n-butyl)phosphate and 1% Octoxynol-9 to inactivate viruses in fresh frozen plasma. Another method for inactivating viruses in blood involves the addition of phenol or formaldehyde to the blood. U.S. Pat. No. 4,833,165. However, both the solvent/detergent method and the phenol/formaldehyde method require removal of the chemical additives prior to clinical use of the blood product.
Inactivation of pathogens in blood products using photoactivated agents has also been described; see, e.g., Wagner et al., Transfusion, 34:521 (1994). However, due to the absorption of light by hemoglobin in several regions in the ultraviolet and visible spectrum, phototreatment is limited in its application to materials containing red blood cells. There is also some indication that phototreatment of red blood cells alters the cells in some manner; see Wagner et al., Transfusion 33:30 (1993).
There is thus a need for compositions and methods for treating blood, blood-derived products, and other biological materials, which will inactivate pathogens present in the products or materials without rendering the products or materials unsuitable for their intended use. Compositions which do not need to be removed from the biological material prior to its use would be particularly useful, as equipment and supplies needed to remove the compositions would be obviated and the costs of handling the biological material would be reduced. This places an additional requirement on the composition, however, in that if the composition remains in the biological material, it must not pose a hazard when the biological material is used for its intended purpose. For example, a highly toxic compound which inactivates pathogens in a blood sample would preclude the use of that blood for transfusion purposes (although the blood sample may still be suitable for laboratory analysis).
It is one intention of this invention to provide compositions and methods of use of the compositions for inactivating pathogens in biological materials, without rendering the materials unsuitable for their intended purpose. Examples of how this may be accomplished include, but are not limited to, using the compounds in an ex vivo or in vitro treatment of the biological materials and then removing the compounds prior to the use of the material; by using a composition which, even though it remains in the material, does not render the material unsuitable for its intended use; or by using a composition which, after inactivating pathogens in the material, will break down to products, where the breakdown products can remain in the material without rendering the material unsuitable for its intended use.
Accordingly, it is an object of this invention to provide compounds for inactivating pathogens in a material, where such compounds comprise a nucleic acid binding moiety; an effector moiety, capable of forming a covalent bond with nucleic acid; and a frangible linker covalently linking the nucleic acid moiety and the effector moiety; wherein the frangible linker degrades so as to no longer covalently link the nucleic acid binding moiety and the effector moiety, under conditions which do not render the material unsuitable for its intended purpose.
It is an additional object of this invention to provide such compounds for inactivating pathogens in a material, wherein the nucleic acid binding moiety is selected from the group consisting of acridine, acridine derivatives, psoralen, isopsoralen and psoralen derivatives.
It is an additional object of this invention to provide such compounds for inactivating pathogens in a material, wherein the frangible linker comprises a functional unit selected from the group consisting of forward esters, reverse esters, forward amides, reverse amides, forward thioesters, reverse thioesters, forward and reverse thionoesters, forward and reverse dithioic acids, sulfates, forward and reverse sulfonates, phosphates, and forward and reverse phosphonate groups, as defined herein.
It is an additional object of this invention to provide such compounds for inactivating pathogens in a material, wherein the effector group comprises a functional unit which is an alkylating agent.
It is an additional object of this invention to provide such compounds for inactivating pathogens in a material, wherein the effector group comprises a functional unit selected from the group consisting of mustard groups, mustard group equivalents, epoxides, aldehydes, and formaldehyde synthons.
It is an additional object of this invention to provide compounds of the formula: 
wherein at least one of R1, R2, R3, R4, R5, R6, R7, R8 and R9 is xe2x80x94Vxe2x80x94Wxe2x80x94Xxe2x80x94E as defined below, and the remainder of R1, R2, R3, R4, R5, R6, R7, R8 and R9 are independently selected from the group consisting of xe2x80x94H, xe2x80x94R10, xe2x80x94Oxe2x80x94R10, xe2x80x94NO2, xe2x80x94NH2, xe2x80x94NHxe2x80x94R10, xe2x80x94N(R10)2, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94C(xe2x95x90O)xe2x80x94R10, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94R10, and xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94R10,
where xe2x80x94R10 is independently H, xe2x80x94C1-8alkyl, xe2x80x94C1-8heteroalkyl, -aryl, -heteroaryl, xe2x80x94C1-3alkyl-aryl, xe2x80x94C1-3heteroalkyl-aryl, xe2x80x94C1-3alkyl-heteroaryl, xe2x80x94C1-3heteroalkyl-heteroaryl, -aryl-C1-3alkyl, -aryl-C1-3heteroalkyl, -heteroaryl-C1-3alkyl, -heteroaryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-aryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl;
V is independently xe2x80x94R11xe2x80x94, xe2x80x94NHxe2x80x94R11xe2x80x94 or xe2x80x94N(CH3)xe2x80x94R11xe2x80x94, where xe2x80x94R11xe2x80x94 is independently xe2x80x94C1-8alkyl-, xe2x80x94C1-8heteroalkyl-, -aryl-, -heteroaryl-, xe2x80x94C1-3alkyl-aryl-, xe2x80x94C1-3heteroalkyl-aryl-, xe2x80x94C1-3alkyl-heteroaryl-, xe2x80x94C1-3heteroalkyl-heteroaryl-, -aryl-C1-3alkyl-, -aryl-C1-3heteroalkyl-, -heteroaryl-C1-3alkyl-, -heteroaryl-C1-3heteroalkyl-, xe2x80x94C1-3alkyl-aryl-C1-3alkyl-, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl-, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl-, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl-, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl-, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl-, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl-, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl-;
W is independently xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94Sxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94Oxe2x80x94, xe2x80x94S(xe2x95x90O)2xe2x80x94Oxe2x80x94, xe2x80x94S(xe2x95x90O)2xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94NR10xe2x80x94, xe2x80x94NR10xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94Oxe2x80x94, xe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94;
X is independently xe2x80x94R11xe2x80x94; and
E is independently selected from the group consisting of xe2x80x94N(R12)2, xe2x80x94N(R12)(R13), xe2x80x94Sxe2x80x94R12, and 
xe2x80x83where xe2x80x94R12 is xe2x80x94CH2CH2xe2x80x94G, where each G is independently xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94CH3, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94CH2xe2x80x94C6H5, or xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94C6H4xe2x80x94CH3;
and where R13 is independently xe2x80x94C1-8alkyl, xe2x80x94C1-8heteroalkyl, -aryl, -heteroaryl, xe2x80x94C1-3alkyl-aryl, xe2x80x94C1-3heteroalkyl-aryl, xe2x80x94C1-3alkyl-heteroaryl, xe2x80x94C1-3heteroalkyl-heteroaryl, xe2x80x94aryl-C1-3alkyl, -aryl-C1-3heteroalkyl, -heteroaryl-C1-3alkyl, -heteroaryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-aryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl;
and all salts and stereoisomers (including enantiomers and diastereomers) thereof.
It is another object of this invention to provide compounds of the formula: 
where R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from the group consisting of xe2x80x94H, xe2x80x94R10, xe2x80x94Oxe2x80x94R10, xe2x80x94NO2, xe2x80x94NH2, xe2x80x94NHxe2x80x94R10, xe2x80x94N(R10)2, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94C(xe2x95x90O)xe2x80x94R10, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94R10, and xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94R10,
where xe2x80x94R10 is independently H, xe2x80x94C1-8alkyl, xe2x80x94C1-8heteroalkyl, -aryl, -heteroaryl, xe2x80x94C1-3alkyl-aryl, xe2x80x94C1-3heteroalkyl-aryl, xe2x80x94C1-3alkyl-heteroaryl, xe2x80x94C1-3heteroalkyl-heteroaryl, -aryl-C1-3alkyl, -aryl-C1-3heteroalkyl, -heteroaryl-C1-3alkyl, -heteroaryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-aryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl;
R20 is xe2x80x94H or xe2x80x94CH3; and
R21, is xe2x80x94R11xe2x80x94Wxe2x80x94Xxe2x80x94E,
where xe2x80x94R11xe2x80x94 is independently xe2x80x94C1-8alkyl-, xe2x80x94C1-8heteroalkyl-, -aryl-, -heteroaryl-, xe2x80x94C1-3alkyl-aryl-, xe2x80x94C1-3heteroalkyl-aryl-, xe2x80x94C1-3alkyl-heteroaryl-, xe2x80x94C3heteroalkyl-heteroaryl-, -aryl-C1-3alkyl-, -aryl-C1-3heteroalkyl-, -heteroaryl-C1-3alkyl-, -heteroaryl-C1-3heteroalkyl-, xe2x80x94C1-3alkyl-aryl-C1-3alkyl-, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl-, xe2x80x94C1-3alkyl-heteroalkyl-, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl-, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl-, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl-, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl-, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl-;
W is independently xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94Sxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94Oxe2x80x94, xe2x80x94S(xe2x95x90O)2xe2x80x94Oxe2x80x94, xe2x80x94S(xe2x95x90O)2xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94NR10xe2x80x94, xe2x80x94NR10xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94Oxe2x80x94, xe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94;
X is independently xe2x80x94R11xe2x80x94; and
E is independently selected from the group consisting of xe2x80x94N(R12)2, xe2x80x94N(R12)(R13), xe2x80x94Sxe2x80x94R12, and 
where xe2x80x94R12 is xe2x80x94CH2CH2xe2x80x94G, where each G is independently xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94CH3, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94CH2xe2x80x94C6H5, or xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94C6H4xe2x80x94CH3;
and where R13 is independently xe2x80x94C1-8alkyl, xe2x80x94C1-8heteroalkyl, -aryl, -heteroaryl, xe2x80x94C1-3alkyl-aryl, xe2x80x94C1-3heteroalkyl-aryl, xe2x80x94C1-3alkyl-heteroaryl, xe2x80x94C1-3heteroalkyl-heteroaryl, -aryl-C1-3alkyl, -aryl-C1-3heteroalkyl, -heteroaryl-C1-3alkyl, -heteroaryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-aryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl;
and all salts and stereoisomers (including enantiomers and diastereomers) thereof.
It is another object of this invention to provide compounds of the formula: 
wherein at least one of R44, R55, R3, R4, R5 and R8 is xe2x80x94Vxe2x80x94Wxe2x80x94Xxe2x80x94E, and the remainder of R44, R55, R3, R4, R5, and R8 are independently selected from the group consisting of xe2x80x94H, xe2x80x94R10, xe2x80x94Oxe2x80x94R10, xe2x80x94NO2, xe2x80x94NH2, xe2x80x94NHxe2x80x94R10, xe2x80x94N(R10)2, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94C(xe2x95x90O)xe2x80x94R10, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94R10, and xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94R10,
where xe2x80x94R10 is independently H, xe2x80x94C1-8alkyl, xe2x80x94C1-8heteroalkyl, -aryl, -heteroaryl, xe2x80x94C1-3alkyl-aryl, xe2x80x94C1-3heteroalkyl-aryl, xe2x80x94C1-3alkyl-heteroaryl, xe2x80x94C1-3heteroalkyl-heteroaryl, -aryl-C1-3alkyl, -aryl-C1-3heteroalkyl, -heteroaryl-C1-3alkyl, -heteroaryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-aryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl;
V is independently xe2x80x94R11xe2x80x94, xe2x80x94NHxe2x80x94R11xe2x80x94 or xe2x80x94N(CH3)xe2x80x94R11xe2x80x94, where xe2x80x94R11xe2x80x94 is independently xe2x80x94C1-8alkyl-, xe2x80x94C1-8heteroalkyl-, -aryl-, -heteroaryl-, xe2x80x94C1-3alkyl-aryl-, xe2x80x94C1-3heteroalkyl-aryl-, xe2x80x94C1-3alkyl-heteroaryl-, xe2x80x94C1-3heteroalkyl-heteroaryl-, -aryl-C1-3alkyl-, -aryl-C1-3heteroalkyl-, -heteroaryl-C1-3alkyl-, -heteroaryl-C1-3heteroalkyl-, xe2x80x94C1-3alkyl-aryl-C1-3alkyl-, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl-, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl-, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl-, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl-, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl-, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl-, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl-;
W is independently xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94Sxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94Oxe2x80x94, xe2x80x94S(xe2x95x90O)2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94NR10xe2x80x94, xe2x80x94NR10xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94Oxe2x80x94, xe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94;
X is independently xe2x80x94R11xe2x80x94, and
E is independently selected from the group consisting of xe2x80x94N(R12)2, xe2x80x94N(R12)(R13), xe2x80x94Sxe2x80x94R12, and 
xe2x80x83where xe2x80x94R12 is xe2x80x94CH2CH2xe2x80x94G, where each G is independently xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94CH3, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94CH2, xe2x80x94C6H5, or xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94C6H4xe2x80x94CH3;
and where R13 is independently xe2x80x94C1-8alkyl, xe2x80x94C1-8heteroalkyl, -aryl, -heteroaryl, xe2x80x94C1-3alkyl-aryl, xe2x80x94C1-3heteroalkyl-aryl, xe2x80x94C1-3alkyl-heteroaryl, xe2x80x94C1-3heteroalkyl-heteroaryl, -aryl-C1-3alkyl, -aryl-C1-3heteroalkyl, -heteroaryl-C1-3alkyl, -heteroaryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-aryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl, xe2x80x94C3alkyl-heteroaryl-C1-3heteroalkyl, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl;
and all salts and stereoisomers (including enantiomers and diastereomers) thereof.
It is yet another object of this invention to provide the compounds xcex2-alanine, N-(2-carbomethoxyacridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester; 4-aminobutyric acid N[(2-carbomethoxyacridin-]-yl), 2-[bis(2-chloroethyl)amino]ethyl ester; 5-aminovaleric acid N-[(2-carbomethoxyacridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester; xcex2-alanine, N-(2-carbomethoxyacridin-]-yl), 3-[bis(2-chloroethyl)amino]propyl ester; xcex2-alanine, [N,N-bis(2-chloroethyl)], 3-[(6-chloro-2-methoxyacridin-9-yl)amino]propyl ester; xcex2-alanine, [N,N-bis(2-chloroethyl)], 2-[(6-chloro-2-methoxyacridin-9-yl)amino]ethyl ester; [N,N-bis(2-chloroethyl)]-2-aminoethyl 4,5xe2x80x2,8-trimethyl-4xe2x80x2-psoralenacetate; and xcex2-alanine, N-(acridin-9-yl), 2-[bis(2-chloroethyl)amino]ethyl ester; and all salts thereof.
Provided are methods for inactivating pathogens in a material, such as a biological material, the methods comprising adding one or more compounds of the invention to the material; and incubating the material. The compound may be added to the material to form a final solution having a concentration of the compound (or total concentration of all compounds, if more than one is used), for example, of between 1 and 500 xcexcM. Biological materials which may be treated include blood, blood products, plasma, platelet preparations, red blood cells, packed red blood cells, serum, cerebrospinal fluid, saliva, urine, sweat, feces, semen, milk, tissue samples, and homogenized tissue samples, derived from human or other mammalian or vertebrate sources.
This invention provides for compounds useful for inactivating pathogens found in materials, particularly for inactivating pathogens found in biological materials such as blood or other body fluids. This invention also provides for methods of use of such compounds for inactivating pathogens in materials. The invention also provides for inactivating pathogens found in or on materials for biological use. The compounds may be used in vitro and ex vivo. The biological materials or materials for biological use may be intended for use in vitro, in vivo, or ex vivo.
The compounds are designed to inactivate pathogens by reacting with nucleic acid. In aqueous solution, at appropriate pH values, the compounds have a period of activity during which they can bind to and react with nucleic acid. After this period, the compounds break down to products which are no longer able to bind to nor react with nucleic acid.
The chemical organization of the compounds can be broadly described as an anchor, covalently bonded to a frangible linker, which is covalently bonded to an effector. xe2x80x9cAnchorxe2x80x9d is defined as a moiety which binds non-covalently to a nucleic acid biopolymer (DNA or RNA). xe2x80x9cEffectorxe2x80x9d is defined as a moiety which reacts with nucleic acid by a mechanism which forms a covalent bond with the nucleic acid. xe2x80x9cFrangible linkerxe2x80x9d is defined as a moiety which serves to covalently link the anchor and effector, and which will degrade under certain conditions so that the anchor and effector are no longer linked covalently. The anchor-frangible linker-effector arrangement enables the compounds to bind specifically to nucleic acid (due to the anchor""s binding ability). This brings the effector into proximity for reaction with the nucleic acid.
The compounds are useful for inactivating pathogens found in materials, particularly biological materials such as blood and other body fluids. Intracellular and extracellular and or other pathogen materials may be inactivated. For example, when a compound of the invention is combined with a pathogen-containing red blood cell composition at physiological pH, the effector portion of the compound reacts with pathogen nucleic acid. Effector moieties which do not react with nucleic acid are gradually hydrolyzed by the solvent. Hydrolysis of the frangible linker occurs concurrently with the effector-nucleic acid reaction and effector hydrolysis. It is desirable that the frangible linker break down at a rate slow enough to permit inactivation of pathogens in the material; that is, the rate of breakdown of the frangible linker is slower than the rate at which the compound reacts with nucleic acid. After a sufficient amount of time has passed, the compound has broken down into the anchor (which may also bear fragments of the frangible linker) and the effector-nucleic acid breakdown products (where fragments of the frangible linker may also remain attached to the effector), or into the anchor (which may also bear fragments of the frangible linker) and the hydrolyzed effector breakdown products (where fragments of the frangible linker may also remain attached to the effector). Additional fragments of the frangible linker may also be generated upon degradation of the compound which do not remain bonded to either the anchor or the effector. The exact embodiment of the compound of the invention determines whether the anchor breakdown product or the effector breakdown product bears fragments of the frangible linker, or whether additional fragments of the frangible linker are generated which do not remain bonded to either the anchor or the effector breakdown products.
A preferred embodiment of the invention comprises compounds which, upon cleavage of the frangible linker, result in breakdown products of low mutagenicity. Mutagenicity of the compounds, after hydrolysis of the effector, is due primarily to the anchor moiety, as the anchor interacts with nucleic acid and may have the potential to interfere with nucleic acid replication, even if the effector moiety has been hydrolyzed. Preferably, after cleavage of the frangible linker, the anchor fragment has substantially reduced mutagenicity.
Definitions
xe2x80x9cPathogenxe2x80x9d is defined as any nucleic acid containing agent capable of causing disease in a human, other mammals, or vertebrates. Examples include microorganisms such as unicellular or multicellular microorganisms. Examples of pathogens are bacteria, viruses, protozoa, fungi, yeasts, molds, and mycoplasmas which cause disease in humans, other mammals, or vertebrates. The genetic material of the pathogen may be DNA or RNA, and the genetic material may be present as single-stranded or double-stranded nucleic acid. The nucleic acid of the pathogen may be in solution, intracellular, extracellular, or bound to cells. Table I lists examples of viruses, and is not intended to limit the invention in any manner.
xe2x80x9cIn vivoxe2x80x9d use of a material or compound is defined as introduction of the material or compound into a living human, mammal, or vertebrate.
xe2x80x9cIn vitroxe2x80x9d use of a material or compound is defined as a use of the material or compound outside a living human, mammal, or vertebrate, where neither the material nor compound is intended for reintroduction into a living human, mammal, or vertebrate. An example of an in vitro use would be the analysis of components of a blood sample using laboratory equipment.
xe2x80x9cEx vivoxe2x80x9d use of a compound is defined as using a compound for treatment of a biological material outside a living human, mammal, or vertebrate, where that treated biological material is intended for use inside a living human, mammal, or vertebrate. For example, removal of blood from a human, and introduction of a compound into that blood to inactivate pathogens, is defined as an ex vivo use of that compound if the blood is intended for reintroduction into that human or another human. Reintroduction of the human blood into that human or another human would be in vivo use of the blood, as opposed to the ex vivo use of the compound. If the compound is still present in the blood when it is reintroduced into the human, then the compound, in addition to its ex vivo use, is also introduced in vivo.
xe2x80x9cBiological materialxe2x80x9d is defined as a material originating from a biological organism of any type. Examples of biological materials include, but are not limited to, blood, blood products such as plasma, platelet preparations, red blood cells, packed red blood cells, and serum, cerebrospinal fluid, saliva, urine, feces, semen, sweat, milk, tissue samples, homogenized tissue samples, and any other substance having its origin in a biological organism. Biological materials also include synthetic material incorporating a substance having its origin in a biological organism, such as a vaccine preparation comprised of alum and a pathogen (the pathogen, in this case, being the substance having its origin in a biological organism), a sample prepared for analysis which is a mixture of blood and analytical reagents, cell culture medium, cell cultures, viral cultures, and other cultures derived from a living organism.
xe2x80x9cMaterial for biological usexe2x80x9d is defined as any material that will come into contact with, or be introduced into, a living human, mammal, or vertebrate, where such contact carries a risk of transmitting disease or pathogens. Such materials include, but are not limited to, medical implants such as pacemakers and artificial joints; implants designed for sustained drug release; needles, intravenous lines, and the like; dental tools; dental materials such as tooth crowns; catheters; and any other material which, when in contact with or introduced into a living human, mammal, or vertebrate, entails risk of transmitting disease or pathogens.
xe2x80x9cInactivation of pathogensxe2x80x9d is defined as rendering pathogens in a material incapable of reproducing. Inactivation is expressed as the negative logarithm of the fraction of remaining pathogens capable of reproducing. Thus, if a compound at a certain concentration renders 99% of the pathogens in a material incapable of reproduction, 1% or one-one hundredth (0.01) of the pathogens remain capable of reproduction. The negative logarithm of 0.01 is 2, and that concentration of that compound is said to have inactivated the pathogens present by 2 logs. Alternatively, the compound is said to have 2 logs kill at that concentration.
xe2x80x9cAlkylxe2x80x9d as used herein refers to a cyclic, branched, or straight chain chemical group containing carbon and hydrogen, such as methyl, pentyl, and adamantyl. Alkyl groups can either be unsubstituted or substituted with one or more substituents, e.g., halogen, alkoxy, acyloxy, amino, hydroxyl, thiol, carboxy, benzyloxy, phenyl, benzyl, or other functionality. Alkyl groups can be saturated or unsaturated (e.g., containing xe2x80x94Cxe2x95x90Cxe2x80x94 or xe2x80x94Cxe2x89xa1xe2x80x94Cxe2x80x94 subunits), at one or several positions. Typically, alkyl groups will comprise 1 to 12 carbon atoms, preferably 1 to 10 carbon atoms, and more preferably 1 to 8 carbon atoms, unless otherwise specified.
xe2x80x9cHeteroalkylxe2x80x9d as used herein are alkyl chains with one or more N, O, S, or P heteroatoms incorporated into the chain. The heteroatom(s) may bear none, one, or more than one of the substituents described above. xe2x80x9cHeteroatomsxe2x80x9d also includes oxidized forms of the heteroatoms N, S and P. Examples of heteroalkyl groups include (but are not limited to) methoxy, ethoxy, and other alkyloxy groups; ether-containing groups; amide containing groups such as polypeptide chains; ring systems such as piperidinyl, lactam and lactone; and other groups which incorporate heteroatoms into the carbon chain. Typically, heteroalkyl groups will comprise, in addition to the heteroatom(s), 1 to 12 carbon atoms, preferably 1 to 10 carbon atoms, and more preferably 1 to 8 carbon atoms, unless otherwise specified.
xe2x80x9cArylxe2x80x9d or xe2x80x9cArxe2x80x9d refers to an unsaturated aromatic carbocyclic group having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl), which can be optionally unsubstituted or substituted with amino, hydroxyl, C1-8alkyl, alkoxy, halo, thiol, and other substituents.
xe2x80x9cHeteroarylxe2x80x9d groups are unsaturated aromatic carbocyclic groups having a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., acridinyl, indolyl or benzothienyl) and having at least one hetero atom, such as N, O, or S, within at least one of the rings. The ring(s) can optionally be unsubstituted or substituted with amino, hydroxyl, alkyl, alkoxy, halo, thiol, acyloxy, carboxy, benzyloxy, phenyl, benzyl, and other substituents.
Abbreviations
The following abbreviations are used: QM (quinacrine mustard); Hct (hematocrit); RBC (red blood cell); LB (Luria Broth); cfu (colony forming units); pfu (plaque forming units); DMEM (Delbecco""s modified eagles medium); FBS (fetal bovine serum); PRBC (packed red blood cells); rpm (revolutions per minute); TC (tissue culture); NHSP (normal human serum pool); NCS (newborn calf serum); PBS (phosphate buffered saline).
Chemical Structure of the Compounds
A wide variety of groups are available for use as the anchors, linkers, and effectors. Examples of anchor groups which can be used in the compound include, but are not limited to, intercalators, minor groove binders, major groove binders, molecules which bind by electrostatic interactions such as polyamines, and molecules which bind by sequence specific interactions. The following is a non-limiting list of possible anchor groups:
acridines (and acridine derivatives, e.g. proflavine, acriflavine, diacridines, acridones, benzacridines, quinacrines), actinomycins, anthracyclinones, rhodomycins, daunomycin, thioxanthenones (and thioxanthenone derivatives, e.g. miracil D), anthramycin, mitomycins, echinomycin (quinomycin A), triostins, ellipticine (and dimers, trimers and analogs thereof), norphilin A, fluorenes (and derivatives, e.g. flourenones, fluorenodiamines), phenazines, phenanthridines, phenothiazines (e.g., chlorpromazine), phenoxazines, benzothiazoles, xanthenes and thioxanthenes, anthraquinones, anthrapyrazoles, benzothiopyranoindoles, 3,4-benzopyrene, 1-pyrenyloxirane, benzanthracenes, benzodipyrones, quinolines (e.g., chloroquine, quinine, phenylquinoline carboxamides), furocoumarins (e.g., psoralens and isopsoralens), ethidium, propidium, coralyne, and polycyclic aromatic hydrocarbons and their oxirane derivatives;
distamycin, netropsin, other lexitropsins, Hoechst 33258 and other Hoechst dyes, DAPI (4xe2x80x2,6-diamidino-2-phenylindole), berenil, and triarylmethane dyes;
aflatoxins;
spermine, spermidine, and other polyamines; and
nucleic acids or analogs which bind by sequence specific interactions such as triple helix formation, D-loop formation, and direct base pairing to single stranded targets. Derivatives of these compounds are also non-limiting examples of anchor groups, where a derivative of a compound includes, but is not limited to, a compound which bears one or more substituent of any type at any location, oxidation or reduction products of the compound, etc.
Examples of linkers which can be used in the invention are, but are not limited to, compounds which include functional groups such as ester (where the carbonyl carbon of the ester is between the anchor and the sp3 oxygen of the ester; this arrangement is also called xe2x80x9cforward esterxe2x80x9d), xe2x80x9creverse esterxe2x80x9d (where the sp3 oxygen of the ester is between the anchor and the carbonyl carbon of the ester), thioester (where the carbonyl carbon of the thioester is between the anchor and the sulfur of the thioester, also called xe2x80x9cforward thioesterxe2x80x9d), reverse thioester (where the sulfur of the thioester is between the anchor and the carbonyl carbon of the thioester, also called xe2x80x9creverse thioesterxe2x80x9d), forward and reverse thionoester, forward and reverse dithioic acid, sulfate, forward and reverse sulfonates, phosphate, and forward and reverse phosphonate groups. xe2x80x9cThioesterxe2x80x9d designates the xe2x80x94C(xe2x95x90O)xe2x80x94Sxe2x80x94 group; xe2x80x9cthionoesterxe2x80x9d designates the xe2x80x94C(xe2x95x90S)xe2x80x94Oxe2x80x94 group, and xe2x80x9cdithioic acidxe2x80x9d designates the xe2x80x94C(xe2x95x90S)xe2x80x94Sxe2x80x94 group. The frangible linker also may include an amide, where the carbonyl carbon of the amide is between the anchor and the nitrogen of the amide (also called a xe2x80x9cforward amidexe2x80x9d), or where the nitrogen of the amide is between the anchor and the carbonyl carbon of the amide (also called a xe2x80x9creverse amidexe2x80x9d). For groups which can be designated as xe2x80x9cforwardxe2x80x9d and xe2x80x9creversexe2x80x9d, the forward orientation is that orientation of the functional groups wherein, after hydrolysis of the functional group, the resulting acidic function is covalently linked to the anchor moiety and the resulting alcohol or thiol function is covalently linked to the effector moiety. The reverse orientation is that orientation of the functional groups wherein, after hydrolysis of the functional group, the resulting acidic function is covalently linked to the effector moiety and the resulting alcohol or thiol function is covalently linked to the anchor moiety.
The frangible linker, such as an amide moiety, also may be capable of degrading under conditions of enzymatic degradation, by endogenous enzymes in the biological material being treated, or by enzymes added to the material.
Examples of effectors which can be used in the invention are, but are not limited to, mustard groups, mustard group equivalents, epoxides, aldehydes, formaldehyde synthons, and other alkylating and cross-linking agents. Mustard groups are defined as including mono or bis haloethylamine groups, and mono haloethylsulfide groups. Mustard group equivalents are defined by groups that react by a mechanism similar to the mustards (that is, by forming an aziridinium intermediate, or by having or by forming an aziridine ring, which can react with a nucleophile), such as mono or bis mesylethylamine groups, mono mesylethylsulfide groups, mono or bis tosylethylamine groups, and mono tosylethylsulfide groups. Formaldehyde synthons are defined as any compound that breaks down to formaldehyde in aqueous solution, including hydroxymethylamines such as hydroxymethylglycine. Examples of formaldehyde synthons are given in U.S. Pat. No. 4,337,269 and in International Patent Application WO 97/02028. While the invention is not limited to any specific mechanism, the effector groups, which are, or are capable of forming an electrophilic group, such as a mustard group, are believed to react with and form a covalent bond to nucleic acid.
Three embodiments of the compounds of this invention are described by the following general formulas I, II, and III.
General formula I is: 
(I)
wherein at least one of R1, R2, R3, R4, R5, R, R7, R8, and R9 is xe2x80x94Vxe2x80x94Wxe2x80x94Xxe2x80x94E as defined below, and the remainder of R1, R2, R3, R4, R5, R6, R7, R8 and R9 are independently selected from the group consisting of xe2x80x94H, xe2x80x94R10, xe2x80x94Oxe2x80x94R10, xe2x80x94NO2, xe2x80x94NH2, xe2x80x94NHxe2x80x94R10, xe2x80x94N(NR10)2, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94C(xe2x95x90O)xe2x80x94R10, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94R10, and xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94R10,
where xe2x80x94R10, is independently H, xe2x80x94C1-8alkyl, xe2x80x94C1-8heteroalkyl, -aryl, -heteroaryl, xe2x80x94C1-3alkyl-aryl, xe2x80x94C1-3heteroalkyl-aryl, xe2x80x94C1-3alkyl-heteroaryl, xe2x80x94C1-3heteroalkyl-heteroaryl, -aryl-C1-3alkyl, -aryl-C1-3heteroalkyl, -heteroaryl-C1-3alkyl, -heteroaryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-aryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl;
V is independently xe2x80x94R11xe2x80x94, xe2x80x94NHxe2x80x94R11xe2x80x94 or xe2x80x94N(CH3)xe2x80x94R11xe2x80x94, where xe2x80x94R11xe2x80x94 is independently xe2x80x94C1-8alkyl-, xe2x80x94C1-8heteroalkyl-, -aryl-, -heteroaryl-, xe2x80x94C1-3alkyl-aryl-, xe2x80x94C1-3heteroalkyl-aryl-, xe2x80x94C1-3alkyl-heteroaryl-, xe2x80x94C1-3heteroalkyl-heteroaryl-, -aryl-C1-3alkyl-, -aryl-C1-3heteroalkyl-, -heteroaryl-C1-3alkyl-, -heteroaryl-C1-3heteroalkyl-, xe2x80x94C1-3alkyl-aryl-C1-3alkyl-, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl-, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl-, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl-, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl-, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl-, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl-, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl-;
W is independently xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94Oxe2x80x94, xe2x80x94S(xe2x95x90O)2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94NR10xe2x80x94, xe2x80x94NR10xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94Oxe2x80x94, xe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94,
X is independently xe2x80x94R11xe2x80x94; and
E is independently selected from the group consisting of xe2x80x94N(R12)2, xe2x80x94N(R12)(R13), xe2x80x94Sxe2x80x94R12, and 
xe2x80x83where xe2x80x94R12 is xe2x80x94CH2CH2xe2x80x94G, where each G is independently xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94CH3, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94CH2xe2x80x94C6H5, or xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94C6H4xe2x80x94CH3;
and where R13 is independently-C1-8alkyl, xe2x80x94C1-8heteroalkyl, -aryl, -heteroaryl, xe2x80x94C1-3alkyl-aryl, xe2x80x94C1-3heteroalkyl-aryl, xe2x80x94C1-3alkyl-heteroaryl, xe2x80x94C1-3heteroalkyl-heteroaryl,-aryl-C1-3alkyl, -aryl-C1-3heteroalkyl, -heteroaryl-C1-3alkyl, -heteroaryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-aryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl;
and all salts and stereoisomers (including enantiomers and diastereomers) thereof.
General formula II is: 
where R1, R2, R3, R4, R5, R6, R7, and R8 are independently selected from the group consisting of xe2x80x94H, xe2x80x94R10, xe2x80x94Oxe2x80x94R10, xe2x80x94NO2, xe2x80x94NH2, xe2x80x94NHxe2x80x94R10, xe2x80x94N(R10)2, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94C(xe2x95x90O)xe2x80x94R10, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94R10, and xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94R10,
where xe2x80x94R10 is independently H, xe2x80x94C1-8alkyl, xe2x80x94C1-8heteroalkyl, -aryl, -heteroaryl, xe2x80x94C1-3alkyl-aryl, xe2x80x94C1-3heteroalkyl-aryl, xe2x80x94C1-3alkyl-heteroaryl, xe2x80x94C1-3heteroalkyl-heteroaryl, -aryl-C1-3alkyl, -aryl-C1-3heteroalkyl, -heteroaryl-C1-3alkyl, -heteroaryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-aryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl;
R20 is xe2x80x94H or xe2x80x94CH3; and
R21 is xe2x80x94R11xe2x80x94Wxe2x80x94Xxe2x80x94E,
where xe2x80x94R11xe2x80x94 is independently xe2x80x94C1-8alkyl-, xe2x80x94C1-8heteroalkyl-, -aryl-, -heteroaryl-, xe2x80x94C1-3alkyl-aryl-, xe2x80x94C1-3heteroalkyl-aryl-, xe2x80x94C1-3alkyl-heteroaryl-, xe2x80x94C1-3heteroalkyl-heteroaryl-, -aryl-C1-3alkyl-, -aryl-C1-3heteroalkyl-, -heteroaryl-C1-3alkyl-, -heteroaryl-C1-3heteroalkyl-, xe2x80x94C1-3alkyl-aryl-C1-3alkyl-, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl-, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl-, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl-, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl-, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl-, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl-, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl-;
W is independently xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94Oxe2x80x94, xe2x80x94S(xe2x95x90O)2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94NR10xe2x80x94, xe2x80x94NR10xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94Oxe2x80x94, xe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94;
X is independently xe2x80x94R11xe2x80x94; and
E is independently selected from the group consisting of xe2x80x94N(R12)2, xe2x80x94N(R12)(R13), xe2x80x94Sxe2x80x94R12, and 
xe2x80x83where xe2x80x94R12 is xe2x80x94CH2CH2xe2x80x94G, where each G is independently xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94CH3, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94CH2xe2x80x94C6H5, or xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94C6H4xe2x80x94CH3;
and where R13 is independently xe2x80x94C1-8alkyl, xe2x80x94C1-8heteroalkyl, -aryl, -heteroaryl, xe2x80x94C1-3alkyl-aryl, xe2x80x94C1-3heteroalkyl-aryl, xe2x80x94C1-3alkyl-heteroaryl, xe2x80x94C1-3heteroalkyl-heteroaryl, -aryl-C1-3alkyl, -aryl-C1-3heteroalkyl, -heteroaryl-C1-3alkyl, -heteroaryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-aryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl;
and all salts and stereoisomers (including enantiomers and diastereomers) thereof.
General formula III is: 
wherein at least one of R44, R55, R3, R4, R5, and R8 is xe2x80x94Vxe2x80x94Wxe2x80x94Xxe2x80x94E, and the remainder of R44, R55, R3, R4, R5, and R8 are independently selected from the group consisting of xe2x80x94H, xe2x80x94R10, xe2x80x94Oxe2x80x94R10, xe2x80x94NO2, xe2x80x94NH2, xe2x80x94NHxe2x80x94R10, xe2x80x94N(R10)2, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94C(xe2x95x90O)xe2x80x94R10, xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94R10, and xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94R10,
where xe2x80x94R10 is independently H, xe2x80x94C1-8alkyl, xe2x80x94C1-8heteroalkyl, -aryl, -heteroaryl, xe2x80x94C1-3alkyl-aryl, xe2x80x94C1-3heteroalkyl-aryl, xe2x80x94C1-3alkyl-heteroaryl, xe2x80x94C1-3heteroalkyl-heteroaryl, -aryl-C1-3alkyl, -aryl-C1-3heteroalkyl, -heteroaryl-C1-3alkyl, -heteroaryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-aryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl;
V is independently xe2x80x94R11xe2x80x94, xe2x80x94NHxe2x80x94R11xe2x80x94 or xe2x80x94N(CH3)xe2x80x94R11xe2x80x94, where xe2x80x94R11xe2x80x94 is independently xe2x80x94C1-8alkyl-, xe2x80x94C1-8heteroalkyl-, -aryl-, -heteroaryl-, xe2x80x94C1-3alkyl-aryl-, xe2x80x94C1-3heteroalkyl-aryl-, xe2x80x94C1-3alkyl-heteroaryl-, xe2x80x94C1-3heteroalkyl-heteroaryl-, -aryl-C1-3alkyl-, -aryl-C1-3heteroalkyl-, -heteroaryl-C1-3alkyl-, -heteroaryl-C1-3heteroalkyl-, xe2x80x94C1-3alkyl-aryl-C1-3alkyl-, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl-, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl-, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl-, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl-, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl-, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl-, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl-;
W is independently xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94Sxe2x80x94, xe2x80x94Sxe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94Oxe2x80x94, xe2x80x94S(xe2x95x90O)2xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94NR10xe2x80x94, xe2x80x94NR10xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94Oxe2x80x94, xe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94Oxe2x80x94, xe2x80x94Oxe2x80x94P(xe2x95x90O)(xe2x80x94OR10)xe2x80x94;
X is independently xe2x80x94R11xe2x80x94; and
E is independently selected from the group consisting of xe2x80x94N(R12)2, xe2x80x94N(R12)(R13), xe2x80x94Sxe2x80x94R12, and 
xe2x80x83where xe2x80x94R12 is xe2x80x94CH2CH2xe2x80x94G, where each G is independently xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94CH3, xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94CH2xe2x80x94C6H5, or xe2x80x94Oxe2x80x94S(xe2x95x90O)2xe2x80x94C6H4xe2x80x94CH3;
and where R13 is independently xe2x80x94C1-8alkyl xe2x80x94C1-8heteroalkyl, -aryl, -heteroaryl, xe2x80x94C1-3alkyl-aryl, xe2x80x94C1-3heteroalkyl-aryl, xe2x80x94C1-3alkyl-heteroaryl, xe2x80x94C1-3heteroalkyl-heteroaryl, -aryl-C1-3alkyl, -aryl-C1-3heteroalkyl, -heteroaryl-C1-3alkyl, -heteroaryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-aryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3alkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3alkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3alkyl, xe2x80x94C1-3heteroalkyl-aryl-C1-3heteroalkyl, xe2x80x94C1-3alkyl-heteroaryl-C1-3heteroalkyl, or xe2x80x94C1-3heteroalkyl-heteroaryl-C1-3heteroalkyl;
and all salts and stereoisomers (including enantiomers and diastereomers) thereof.
It will be appreciated that, in general formula I above, the acridine nucleus is the anchor moiety, the xe2x80x94Vxe2x80x94Wxe2x80x94Xxe2x80x94 group(s) comprises the frangible linker, and the E group(s) is the effector group. Similarly, in general formula III above, the psoralen nucleus is the anchor moiety, the xe2x80x94Vxe2x80x94Wxe2x80x94Xxe2x80x94 group(s) comprises the frangible linker, and the E group(s) is the effector group. General formula II is a subset of general formula I.
An exemplary compound of the invention is the structure below, designated IV: 
In IV, a 2-carbomethoxyacridine ring system serves as the anchor moiety via intercalation. A bis (chloroethyl) amine group serves as the effector moiety, which can alkylate nucleic acid; the nitrogen mustard hydrolyzes if it does not react with nucleic acid. The linker is xe2x80x94NHxe2x80x94CH2CH2xe2x80x94C(xe2x95x90O)xe2x80x94Oxe2x80x94CH2CH2xe2x80x94. In aqueous solution at physiological pH, this ester-containing linker hydrolyzes within hours. Changing the pH of the solution changes the rate at which the linker hydrolyzes; for the corresponding alcohol analog of IV (where the xe2x80x94Cl atoms of IV are replaced with xe2x80x94OH groups), xe2x89xa61% hydrolysis of the ester linkage is observed at pH 3 after 100 minutes at 37xc2x0 C.; at pH 8, more than 50% hydrolysis of the ester linkage is observed after 100 minutes at 37xc2x0 C. The resulting hydrolysis products of IV are N-(2-carbomethoxy-9-acridinyl)-xcex2-alanine and triethanolamine: 
where the 2-carbomethoxyacridine bears xcex2-alanine as a linker fragment, and the effector breakdown product bears an ethanol group as a linker fragment.
At physiological pH values, the carboxylate of the xcex2-alanine will be negatively charged, a feature which decreases the tendency of the attached 2-carbomethoxyacridine group to intercalate into a negatively charged nucleic acid molecule. This lowers the mutagenicity of N-(2-carbomethoxy-9-acridinyl)-xcex2-alanine relative to 9-aminoacridine. This potential for lowering the mutagenicity of the anchor fragment illustrates one advantage provided by the frangible linker.
Another advantage of the frangible linker in compounds similar to IV is that the hydrolysis rate can be adjusted by varying the length of the linker arm between the 9-aminoacridine anchor moiety and the ester function. As described in Example 7 and Tables III and IV below, an increase in the number of methylene groups between the aminoacridine anchor and the ester group results in a decrease in the amount of hydrolysis seen in aqueous solution, at pH 8, 37xc2x0 C., for diol analogs of certain compounds of the invention (where the xe2x80x94Cl atoms of the mustards are replaced with xe2x80x94OH groups).
Examples of the compounds of the invention are given below, as illustration and not as any limitation on the invention. 
Applications
Examples of uses of the compounds of the invention include, but are not limited to: addition of the compounds of the invention in solid or solution form to biological materials, to inactivate pathogens present in the biological materials; immersion or other treatment of a material for biological use in a solution of the compounds of the invention, to inactivate pathogens present in or on the material; and inclusion of compounds of the invention in targeted liposomes, to direct the compounds to particular cells in order to damage the nucleic acid of those cells.
It should be noted that while the compounds of the invention are designed to hydrolyze under certain conditions, they are stable under other conditions. It is desirable for the frangible linker and the effector group(s) to be relatively stable under certain conditions used for storage. Examples of manners in which the compounds may be stored include, but are not limited to, dry solids, oils with low water content, frozen aqueous solutions, frozen non-aqueous solutions, suspensions, and solutions which do not permit hydrolysis of the frangible linker or the effector group(s), for example liquid non-aqueous solutions. The compounds may be stored at temperatures at or below 0xc2x0 C. (e.g., in a freezer), or at temperatures above 0xc2x0 C. (e.g., in a refrigerator or at ambient temperatures). The compounds preferably are stable under the storage conditions for a period of between three days and one year, between one week and one year, between one month and one year, between three months and one year, between six months and one year, between one week and six months, between one month and six months, between three months and six months, between one week and three months, or between one month and three months. The stability of the compounds will be determined both by the temperature at which they are stored, and by the state in which they are stored (e.g., non-aqueous solution, dry solid).
Conditions for Pathogen Inactivation
Conditions for treating biological materials with a pathogen inactivating compound may be selected based on the selected material and the inactivating compound. Typical concentrations of pathogen inactivating compound for the treatment of biological materials such as blood products are on the order of about 0.1 xcexcM to 5 mM, for example about 500 xcexcM. For example, a concentration of pathogen inactivating compound may be used which is sufficient to inactivate at least about 1 log, or at least about 2 logs, or for example, at least about 3 to 6 logs of a pathogen in the sample. In one embodiment, the pathogen inactivating compound produces at least 1 log kill at a concentration of no greater than about 500 xcexcM, more preferably at least 3 logs kill at no greater than 500 xcexcM concentration. In another non-limiting example, the pathogen inactivating compound may have at least 1 log kill, and preferably at least 6 logs kill at a concentration of about 0.1 xcexcM to about 3 mM.
Incubation of blood products with the pathogen inactivating compound can be conducted for example, for about 5 minutes to 72 hours or more, or about 1 to 48 hours, for example, about 1 to 24 hours, or, for example, about 8 to 20 hours. For red blood cells, the incubation is typically conducted at a temperature of about 2xc2x0 C. to 37xc2x0 C., preferably about 18xc2x0 C. to 25xc2x0 C. For platelets, the temperature is preferably about 20 to 24xc2x0 C. For plasma, the temperature may be about 0 to 60xc2x0 C., typically about 0-24xc2x0 C. The pH of the material being treated is preferably about 4 to 10, more preferably about 6 to 8.
One embodiment of the invention encompasses compounds and methods for use in inactivating pathogens in blood or blood products, and a preferred set of storage conditions for this purpose would be those conditions that allow the convenient storage and use of the compounds at blood banks.
Under the conditions used for pathogen inactivation in or on a material, the frangible linker and effector group(s) will undergo hydrolysis or reaction. The hydrolysis, of both the frangible linker and the effector groups(s), preferably is slow enough to enable the desired amount of pathogen inactivation to take place. The time required for pathogen inactivation may be, for example, about 5 minutes to 72 hours.
Treatment of Red Blood Cells
Preferably, treatment of red blood cell containing materials with the pathogen inactivating compound does not damage red blood cell function or modify red blood cells after treatment. The lack of a substantially damaging effect on red blood cell function may be measured by methods known in the art for testing red blood cell function. For example, the levels of indicators such as intracellular ATP (adenosine 5xe2x80x2-triphosphate), intracellular 2,3-DPG (2,3-diphosphoglycerol) or extracellular potassium may be measured, and compared to an untreated control. Additionally hemolysis, pH, hematocrit, hemoglobin, osmotic fragility, glucose consumption and lactate production may be measured.
Methods for determining ATP, 2,3-DPG, glucose, hemoglobin, hemolysis, and potassium are available in the art. See for example, Davey et al., Transfusion, 32:525-528 (1992), the disclosure of which is incorporated herein. Methods for determining red blood cell function are also described in Greenwalt et al., Vox Sang, 58:94-99 (1990); Hogman et al., Vox Sang, 65:271-278 (1993); and Beutler et al., Blood, Vol. 59 (1982) the disclosures of which are incorporated herein by reference. Extracellular potassium levels may be measured using a Ciba Coming Model 614 K+/Na+ Analyzer (Ciba Coming Diagnostics Corp., Medford, Mass.). The pH can be measured using a Ciba Coming Model 238 Blood Gas Analyzer (Ciba Coming Diagnostics Corp., Medford, Mass.).
Binding of species such as IgG, albumin, and IgM to red blood cells also may be measured using methods available in the art. Binding of molecules to red blood cells can be detected using antibodies, for example to acridine and IgG. Antibodies for use in assays can be obtained commercially, or can be made using methods available in the art, for example as described in Harlow and Lane, xe2x80x9cAntibodies, a Laboratory Manual, Cold Spring Harbor Laboratory,xe2x80x9d 1988, the disclosure of which is incorporated herein. For example, anti-IgG is commercially available from Caltag, Burlingame, Calif.; Sigma Chemical Co., St. Louis, Mo. and Lampire Biological Laboratory, Pipersvelle, Pa.
In a method of treatment of a material comprising red blood cells with the pathogen inactivating compound, preferably the level of extracellular potassium is not greater than 3 times, more preferably no more than 2 times the amount exhibited in the untreated control after 1 day. In another embodiment, preferably, hemolysis of the treated red blood cells is less than 3% after 28 day storage, more preferably less than 2% after 42 day storage, and most preferably less than or equal to about 1% after 42 day storage at 4xc2x0 C.
Biological Materials
A variety of biological materials may be treated with a pathogen inactivating compound. Biological materials include blood products such as whole blood, packed red blood cells, platelets and fresh or frozen plasma. Blood products further encompass plasma protein portion, antihemophilic factor (Factor VIII), Factor IX and Factor IX complex, fibrinogens, Factor XIII, prothrombin and thrombin, immunoglobulins (such as IgG, IgA, IgD, IgE and IgM and fragments thereof), albumin, interferon, and lymphokines. Also contemplated are synthetic blood products.
Other biological materials include vaccines, recombinant DNA produced proteins and oligopeptide ligands. Also encompassed are clinical samples such as urine, sweat, sputum, feces, spinal fluid. Further encompassed are synthetic blood or blood product storage media.
Reducing the Concentration of Compounds in Materials after Treatment
The concentration of the pathogen inactivating compound in a biological material, such as a blood product, can be reduced after the treatment. Methods and devices which may be used are described in PCT/US96/09846; U.S. Ser. No. 08/779,830, filed Jan. 6, 1997; and in the co-filed application, xe2x80x9cMethods and Devices for the Reduction of Small Organic Compounds from Blood Productsxe2x80x9d, PCT/US98/00531, filed Jan. 6, 1998, the disclosures of each of which are incorporated herein by reference in their entirety.
Quenching
In another embodiment the compounds of the invention may be used in combination with a quencher. Methods for quenching undesired side reactions of pathogen inactivating compounds in biological materials are described in the cofiled U.S. Provisional Application Serial No. 60/070,597, filed Jan. 6, 1998, Attorney Docket No. 282173000600, xe2x80x9cMethods for Quenching Pathogen Inactivators in Biological Materials,xe2x80x9d the disclosure of which is incorporated herein. Disclosed in the cofiled application are methods for quenching undesired side reactions of a pathogen inactivating compound that includes a functional group which is, or which is capable of forming, an electrophilic group. In this embodiment, the material is treated with the pathogen inactivating compound and a quencher, wherein the quencher comprises a nucleophilic functional group that is capable of covalently reacting with the electrophilic group. Preferred quenchers are thiols, such as glutathione.